[Correlation analysis between abundance of K-ras mutation in plasma free DNA and its correlation with clinical outcome and prognosis in patients with metastatic colorectal cancer].

OBJECTIVE: To detect K-ras gene mutations in plasma free DNA by peptide nucleic acid clamp PCR assay (PNA-PCR) and nested primer PCR, and to analyze the correlation between K-ras mutations and prognosis in patients with metastatic colorectal cancer (mCRC). METHODS: Peripheral blood was collected and free DNA was extracted from plasma in 106 patients with mCRC. Nested primer PCR and PNA-PCR were used to detect K-ras gene mutation in the plasma free DNA. The patients were divided into three groups by K-ras status: wild-type group (wild-type determined by both methods), low mutation group (mutation by PNA-PCR method, wild-type by nested primer PCR method) and high mutation group (mutation by two methods). The correlation between K-ras mutations and prognosis was analyzed. RESULTS: The mutation rate of K-ras in tumor tissues of the 106 patients was 40.6%. The Mutation rate of K-ras in plasma free DNA detected by PNA-PCR was 31.1%, significantly higher than that of 15.1% detected by nested primer PCR (P = 0.006). The consistent rate of the K-ras status in plasma free DNA detected by PNA-PCR and that in tumor tissue detected by traditional method was up to 83.0%. The median overall survival (OS) of patients of the wild type, low mutation and high mutation groups was 23.5 months, 17.3 months and 13.9 months, respectively (P = 0.002). The median progression-free survival (PFS) of the K-ras wild-type, low mutation and high mutation groups with first-line chemotherapy was 6.8 months, 6.1 months and 3.2 months, respectively (P = 0.002), and the median OS of them were 23.0 months, 15.5 months and 13.9 months, respectively (P = 0.036). The overall response rate (ORR) was improved in the K-ras wide-type patients who received cetuximab combined with chemotherapy as first-line therapy (75.0% vs. 23.4%, P = 0.058). Cetuximab combined with in second-line therapy chemotherapy led to a significant improvement in disease control rate (DCR) ( 100% vs. 35.7%, P < 0.001) as compared with those of chemotherapy alone. COX regression model showed that K-ras status detected by PNA-PCR, ECOG PS, number of surgery and initially metastatic site were independent factors for prognosis. CONCLUSIONS: PNA-PCR for the detection of K-ras mutation in plasma free DNA can be used to substitute the traditional method for detection of K-ras mutation in tumor tissues. The abundance of K-ras mutation in plasma free DNA is an independent prognostic factor for patients with metastatic colorectal cancer.

[Analysis of therapeutic effect and prognosis in patients with metastatic colorectal cancer and different K-ras status].

OBJECTIVE: To evaluate the correlation of clinical effect and prognosis between patients with metastatic colorectal cancer (mCRC) and different K-ras status. METHODS: The clinical characteristics, chemotherapeutic regimens and survival of 153 mCRC patients with different K-ras status were analyzed retrospectively. RESULTS: The median overall survival (OS) in patients without K-ras mutation were 31.7 months, significantly longer than 21.3 months in the patients with K-ras mutation (P = 0.037). The median progression-free survival (PFS) and OS in patients who received chemotherapy followed by anti-EGFR antibody treatment were 11.5 and 39.3 months, respectively, significantly longer as compared with the PFS and OS in those received chemotherapy in combination with anti-EGFR antibody concomitantly (5.7, P = 0.02, and 28.7 months, P = 0.034, respectively). CONCLUSIONS: K-ras status is a prognostic biomarker for mCRC patients treated with anti-EGFR antibody. The combination settings of anti-EGFR in combination with chemotherapy may improve survival of mCRC patients with wild-type K-ras status.

Physiological Function of RDL1 and RDL2 Subunits of the Ionotropic GABA Receptor in the Spodoptera litura with the CRISPR/Cas9 System In Vivo.

In insect ionotropic γ-aminobutyric acid receptor (iGABAR) subunits, only resistance to dieldrin (RDL) can be individually and functionally expressed in vitro. In lepidopteran, two to three RDL subtypes are identified; however, their physiological roles have not been distinguished in vivo. In this study, SlRdl1 and SlRdl2 of S. litura were individually knocked out using CRISPR/Cas9, respectively. The mortality and larval and pupal duration of KOSlRdl1 and KOSlRdl2 were increased. The flight time and distance were increased by 43.30%-80.66% and 58.96%-198.22%, respectively, in KOSlRdl1. The GABA-induced current was significantly decreased by 53.57%-74.28% and 46.91%-63.34% in the ventral nerve cord, and the GABA titer was significantly reduced by 17.65%-28.05% and 19.85%-42.46% in KOSlRdl1 and KOSlRdl2, respectively. In conclusion, SlRdl1 and SlRdl2 are necessary for the transmission of GABA-induced neural signals; however, only SlRdl1 could regulate the flight capability of S. litura. Our results provided a new avenue to study lepidopteran iGABARs.

Expression and prognostic value of VEGFR-2, PDGFR-ß, and c-Met in advanced hepatocellular carcinoma.

BACKGROUND: We explore the clinical and prognostic significance of expression of vascular endothelial growth factor receptor (VEGFR)-2, platelet-derived growth factor receptor (PDGFR)-ß, and c-Met in patients with hepatocellular carcinoma (HCC). METHODS: The expression of VEGFR-2, PDGFR-ß, and c-Met were determined by immunohistochemical examination of the tissues of 93 HCC patients. The relationships of these markers with clinicopathological factors and prognosis were then analyzed. RESULTS: High expression of VEGFR-2, PDGFR-ß, and c-Met was found in 86%, 19.4%, and 80.6% of patients, respectively. Expression of VEGFR-2 correlated with gender (P = 0.044), hepatitis B surface antigen positivity (P = 0.024), degree of tumor differentiation (P = 0.023), and hepatic cirrhosis (P = 0.026). Expression of PDGFR-ß correlated with alpha-fetoprotein level (P = 0.029), tumor size (P = 0.033), and hepatic cirrhosis (P = 0.023). No significant correlations were identified between expression of c-Met and clinicopathological factors. Expression of PDGFR-ß correlated with overall survival (P = 0.046) and expression of c-Met correlated with progression-free survival (P = 0.01). CONCLUSIONS: We found that in patients with HCC, high expression of VEGFR-2 correlates with chronic hepatitis B virus infection and hepatic cirrhosis. High expression of PDGFR-ß is a predictor of poor prognosis. High expression of C-Met may predict therapeutic effectiveness of sorafenib in HCC patients.

[Expression of Osterix mRNA and protein levels in cultured human periodontal ligament cells after mechanical stimulation].

OBJECTIVE: To examine the expression of Osterix (Osx) mRNA and protein after application of mechanical force on human periodontal ligament cells (HPDLCs), and to investigate the role of Osx in orthodontic alveolar bone remodeling. METHODS: HPDLCs were isolated and cultured in vitro with explant method. Approximately 2.5 x 10(5) cells were seeded onto six-well cell culture plates and then were exposed to centrifugal force for 1, 2, 4, 6, 8 or 12 h at 631 r x min(-1). The expression of Osx mRNA and protein was measured by reverse transcription-polymerase chain raction (RT-PCR) and Western blot respectively. Immunofluorescence assay was used to detect the expression and subcellular At the initial time point, Osx mRNA had a weak exlocalization of Osx protein by green fluorescence. RESULTS: pression and protein was not detected. Under the mechanical stimulation, both mRNA and protein levels of Osx were upregulated in a time-dependent manner. Furthermore, Osx protein was translocated gradually from the cytosol into the cell nuclei. CONCLUSION: The expression and activation of Osx were enhanced by mechanical stress in HPDLCs, which indicates that Osx may play an important role in HPDLCs osteogenic differentiation and periodontal tissue remodeling induced by mechanical stress.

[Changes of mRNA and protein levels of activating transcription factor-4 after centrifugal force stimulation in cultured human periodontal ligament cells].

PURPOSE: This study was designed to examine the changes of mRNA and protein levels of activating transcription factor-4 after centrifugal force stimulation in cultured human periodontal ligament (PDL)cells. METHODS: Human PDL cells were cultured by sequential digestio, and then stimulated by centrifugal force (167g) for 10, 30, 60, 90, 120 and 240 minutes. The changes of mRNA and protein levels of ATF4 were analyzed by semi-quantitative RT-PCR and Western blotting. The data was analyzed with SPSS11.5 software package for ANOVA. RESULTS: Unstimulated human PDL cells exhibit an ATF4 mRNA expression, but very low level of protein; Immediately 10 minutes after force application, the mRNA and protein levels of ATF4 increased(P>0.05); at 30-minute,the mRNA level increased significantly to a peak level(P<0.01), at 60-minute, the protein level increased significantly to a peak level(P<0.01); at 90-minute, mRNA and protein levels decreased, but still higher than the control(P<0.01); after 120 minutes, mRNA and protein levels decreased to control levels(P>0.05). CONCLUSION: Centrifugal force stimulates ATF4 gene expression within a narrow time frame: a temporary increase of ATF4 expression and a fast downregulation back to its preloading level. This study demonstrates that ATF4 may play a critical role in orthodontic alveolar bone remodeling.

Melissoidesin G, a diterpenoid purified from Isodon melissoides, induces leukemic-cell apoptosis through induction of redox imbalance and exhibits synergy with other anticancer agents.

Melissoidesin G (MOG) is a new diterpenoid purified from Isodon melissoides, a plant used in Chinese traditional medicine as antitumor and anti-inflammatory agents. In our study, MOG was shown to specifically inhibit the growth of human leukemia cell lines and primary acute myeloid leukemia (AML) blasts via induction of apoptosis, with the evidence of mitochondrial DeltaPsim loss, reactive oxygen species production, caspases activation and nuclear fragmentation. Furthermore, it was shown that thiol-containing antioxidants completely blocked MOG-induced mitochondrial DeltaPsim loss and subsequent cell apoptosis, while the inhibition of apoptosis by benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone only partially attenuated mitochondrial DeltaPsim loss, indicating that MOG-induced redox imbalance is an early event upstream to mitochondrial DeltaPsim loss and caspase-3 activation. Consistently, it was found that MOG rapidly decreased the intracellular glutathione (GSH) content in a dose-dependent manner and the significance of GSH depletion in MOG-induced apoptosis was further supported by the protective effects of tert-butylhydroquinone (tBHQ) and the facilitative effects of DL-buthionine (S,R)-sulfoximine (BSO). Furthermore, it was showed that GSH depletion induced by MOG rendered some leukemia cell lines more sensitive to arsenic trioxide (As2O3), doxorubicin or cisplatin. Additionally, the synergistic apoptotic effects of MOG with As2O3 were detected in HL-60 and primary AML cells, but not in normal cells, suggesting the selective toxicity of their combination to the malignant cells. Together, we proposed that MOG alone or administered with other anticancer agents may provide a novel therapeutic strategy for leukemia.